摘要
目的 表达及纯化人黑素浓集素受体1抗原表位肽,探讨其在白癜风患者自身抗体检测中的应用.方法 人黑素浓集素受体1抗原表位肽编码基因合成后克隆至原核表达载体pGEX-4T-2,转化大肠杆菌BL21菌株,异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析和Western印迹鉴定目的 蛋白表达.以提取的黑素细胞膜蛋白作为阻断抗原,进行阻断ELISA分析.以目的 蛋白为抗原,检测100例进展期白癜风患者和30例正常人血清IgG抗体情况.结果 成功构建重组表达载体,重组蛋白成功表达并纯化,上样量低于0.0625μg时纯化率达100%.目的 蛋白与白癜风患者血清IgG抗体间的结合能被天然黑素细胞膜抗原抑制.100例进展期白癜风患者血清中有36例与目的蛋白结合反应呈阳性.结论 成功表达并纯化了人黑素浓集素受体1抗原表位肽,该目的蛋白能够结合白癜风患者血清中IgG抗体,可以用于抗人黑素浓集素受体1抗体的检测.
Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside （IPTG） and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase （GST） purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2010年第12期851-854,共4页
Chinese Journal of Dermatology
基金
国家自然科学基金（30872280,30800563）
卫生部科研基金（WKJ2008-2-033）
关键词
白癜风
黑素浓集素受体1
克隆
分子
Vitiligo
Melanin-concentrating hormone receptor 1
Cloning, molecular
